Conserved Local Structural Motifs in Glycoside Hydrolase Families Facilitate the Discovery of Functional Enzymes

Glycoside hydrolases (GHs) are vital for natural glycoside biotransformation, especially in enhancing the pharmacological effects of natural products like ginsenosides. In this study, we collected 67 microbial-derived ginsenoside-hydrolyzing enzymes from nine GH families. Despite differences in global structures, the key residues surrounding substrate binding in GH1 and GH3 exhibit conserved structural motifs. Leveraging these motifs, five GH genes from Cellulosimicrobium were cloned, and three enzymes (Cbgl496, Cbgl516, Cbgl766) were characterized. Experimental results demonstrated that Cbgl516, Cbgl766, and Cbgl841 specifically catalyzed the hydrolysis of the β(1–6) glycosidic bond in the C-20 sugar chain of ginsenoside Rb1 to yield Rd. Cbgl496 selectively catalyzed the hydrolysis of β(1–2) glycosidic bonds in the oligosaccharide chains at the C-3 position of ginsenosides Rb1, Rb2, Rb3, and Rc, thereby directionally producing the minor ginsenosides Gy XVII, Compound O, Compound Mx1, and Compound Mc1. Structural analysis of 109,994 GH1/GH3 models from AlphaFold database revealed conserved residues across various organisms, emphasizing evolutionary conservation in the 3D structure of the catalytic core region despite sequence diversity. This study underscores the importance of conserved local structural motifs in GHs, offering insights for functional enzyme screening and understanding enzyme diversity and industrial applications.